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Cooperative regulation of Ca(v)1.2 channels by intracellular Mg(2+), the proximal C-terminal EF-hand, and the distal C-terminal domain.

Brunet S, Scheuer T, Catterall WA - J. Gen. Physiol. (2009)

Bottom Line: We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion.The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue.Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. sbrunet@u.washington.edu

ABSTRACT
L-type Ca(2+) currents conducted by Ca(v)1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular Mg(2+) (Mg(i)) inhibits the ionic current of Ca(v)1.2 channels. Because Mg(i) is altered in ischemia and heart failure, its regulation of Ca(v)1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg(i) on voltage-dependent inactivation (VDI) of Ca(v)1.2 channels using Na(+) as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca(2+)-dependent inactivation process. We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion. The increased rate and extent of VDI caused by increased Mg(i) were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg(i) to the EF-hand (K(D) approximately 0.9 mM) near the resting level of Mg(i) in ventricular myocytes. VDI was more rapid for L-type Ca(2+) currents in ventricular myocytes than for Ca(v)1.2 channels in transfected cells. Coexpression of Ca(v)beta(2b) subunits and formation of an autoinhibitory complex of truncated Ca(v)1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca(v)1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

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Mgi modulation of the voltage dependence of inactivation of WT CaV1.2 and C-terminal EF-hand mutant (CaV1.2(D1546K)) with Na+ as charge carrier. Comparison of mean inactivation–voltage relationships for ICaV1.2(Na) of WT CaV1.2 (A) and C-terminal EF-hand mutant (D1546K) (B) exposed to the indicated Mgi. (C) Plot of fraction Inon-inact versus log [Mgi] for WT CaV1.2 and C-terminal EF-hand mutant (D1546K). Curves represent the best fit of a binding isotherm to the data, with KD = 0.78 ± 0.04 mM for WT and KD = 3.6 ± 1.4 mM Mgi for D1546K.
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fig5: Mgi modulation of the voltage dependence of inactivation of WT CaV1.2 and C-terminal EF-hand mutant (CaV1.2(D1546K)) with Na+ as charge carrier. Comparison of mean inactivation–voltage relationships for ICaV1.2(Na) of WT CaV1.2 (A) and C-terminal EF-hand mutant (D1546K) (B) exposed to the indicated Mgi. (C) Plot of fraction Inon-inact versus log [Mgi] for WT CaV1.2 and C-terminal EF-hand mutant (D1546K). Curves represent the best fit of a binding isotherm to the data, with KD = 0.78 ± 0.04 mM for WT and KD = 3.6 ± 1.4 mM Mgi for D1546K.

Mentions: To further explore the role of Mgi and the EF-hand in modulation of VDI, we studied steady-state inactivation of WT CaV1.2 or EF-hand mutant (D1546K) channels expressed in tsA-201 cells. For WT CaV1.2 channels at low Mgi concentration, steady-state inactivation was incomplete compared with higher Mgi concentrations (Fig. 5 A and Table I). Elevation of Mgi resulted in a negative shift in the voltage dependence of inactivation and an increase of maximal inactivation at positive potentials (Fig. 5 A). These effects are similar to those observed when Ba2+ was used as charge carrier for transfected CaV1.2 channels (Brunet et al., 2005b) and for myocyte L-type Ca2+ currents (Hartzell and White, 1989). Complete inactivation at depolarized potentials was observed with 2.4 and 7.2 mM Mgi in these experiments using Na+ as a charge carrier (Fig. 5 A and Table I), but not when Ba2+ was the permeant ion (Brunet et al., 2005b).


Cooperative regulation of Ca(v)1.2 channels by intracellular Mg(2+), the proximal C-terminal EF-hand, and the distal C-terminal domain.

Brunet S, Scheuer T, Catterall WA - J. Gen. Physiol. (2009)

Mgi modulation of the voltage dependence of inactivation of WT CaV1.2 and C-terminal EF-hand mutant (CaV1.2(D1546K)) with Na+ as charge carrier. Comparison of mean inactivation–voltage relationships for ICaV1.2(Na) of WT CaV1.2 (A) and C-terminal EF-hand mutant (D1546K) (B) exposed to the indicated Mgi. (C) Plot of fraction Inon-inact versus log [Mgi] for WT CaV1.2 and C-terminal EF-hand mutant (D1546K). Curves represent the best fit of a binding isotherm to the data, with KD = 0.78 ± 0.04 mM for WT and KD = 3.6 ± 1.4 mM Mgi for D1546K.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2717695&req=5

fig5: Mgi modulation of the voltage dependence of inactivation of WT CaV1.2 and C-terminal EF-hand mutant (CaV1.2(D1546K)) with Na+ as charge carrier. Comparison of mean inactivation–voltage relationships for ICaV1.2(Na) of WT CaV1.2 (A) and C-terminal EF-hand mutant (D1546K) (B) exposed to the indicated Mgi. (C) Plot of fraction Inon-inact versus log [Mgi] for WT CaV1.2 and C-terminal EF-hand mutant (D1546K). Curves represent the best fit of a binding isotherm to the data, with KD = 0.78 ± 0.04 mM for WT and KD = 3.6 ± 1.4 mM Mgi for D1546K.
Mentions: To further explore the role of Mgi and the EF-hand in modulation of VDI, we studied steady-state inactivation of WT CaV1.2 or EF-hand mutant (D1546K) channels expressed in tsA-201 cells. For WT CaV1.2 channels at low Mgi concentration, steady-state inactivation was incomplete compared with higher Mgi concentrations (Fig. 5 A and Table I). Elevation of Mgi resulted in a negative shift in the voltage dependence of inactivation and an increase of maximal inactivation at positive potentials (Fig. 5 A). These effects are similar to those observed when Ba2+ was used as charge carrier for transfected CaV1.2 channels (Brunet et al., 2005b) and for myocyte L-type Ca2+ currents (Hartzell and White, 1989). Complete inactivation at depolarized potentials was observed with 2.4 and 7.2 mM Mgi in these experiments using Na+ as a charge carrier (Fig. 5 A and Table I), but not when Ba2+ was the permeant ion (Brunet et al., 2005b).

Bottom Line: We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion.The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue.Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, WA 98195, USA. sbrunet@u.washington.edu

ABSTRACT
L-type Ca(2+) currents conducted by Ca(v)1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular Mg(2+) (Mg(i)) inhibits the ionic current of Ca(v)1.2 channels. Because Mg(i) is altered in ischemia and heart failure, its regulation of Ca(v)1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg(i) on voltage-dependent inactivation (VDI) of Ca(v)1.2 channels using Na(+) as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca(2+)-dependent inactivation process. We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion. The increased rate and extent of VDI caused by increased Mg(i) were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg(i) to the EF-hand (K(D) approximately 0.9 mM) near the resting level of Mg(i) in ventricular myocytes. VDI was more rapid for L-type Ca(2+) currents in ventricular myocytes than for Ca(v)1.2 channels in transfected cells. Coexpression of Ca(v)beta(2b) subunits and formation of an autoinhibitory complex of truncated Ca(v)1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca(v)1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

Show MeSH
Related in: MedlinePlus